Human monoclonal antibodies (Hu-MAbs) from lymphocytes of melanoma patients have identified a new group of apparently cytoplasmic tumor-associated antigens. These Hu-MADAs are not differentiation antigens of neural crest cells or obvious structural components of melanoma cells and are found in tumors of unrelated lineages. Most importantly, they are detected in tumors and not in normal tissues of the same histology. Because of their unique distribution, they are of theoretical and practical interest. Hu-MADAs defined by 4 IgG Hu-MAbs from 3 patients are proteins by several criteria. HPLC and standard biochemical methods will be combined with affinity chromatography to identify the cytosol and membrane components of melanoma cells that certain antigenic activity. The membrane location will be verified by subcellular fractionation. The relationship of the 2 fractions and their biosynthesis and processing will be examined by pulse-chase analysis. Recombinant DNA technology will be used as a complementary approach to directly determine the identity of these antigens to oncogene products. The 2 lambda gt11 cDNA libraries we have constructed will be immunologically screened in situ for positive clones. Unique positive clones confirmed by mapping with restriction enzymes and DNA hybridization will be sequenced for comparison with existing data banks in the BIONET network. The amino acid specificity of the kinase activity associated with the immune precipitate of 10-3-44 will be examined. The precursor-product relationship of p220 and p125 will be studied by Cleveland gel analysis. The usefulness of the Hu-MAbs in dermatopathological diagnosis will be studied. Their selective reactivity to melanomas and not nevus cells will be tested in 30 benign lesions with cytologic atypia (epithelioid nevi from children). Thirty cases of melanoma associated with preexisting nevi will be stained to determine in the Hu-MADAs might aid in measuring depth of melanoma in situ. Their expression to see if Hu-MAbs might identify patients truly at risk for the development of melanoma. The detectability of Hu-MAbs in samples of vitreous humor from 30 patients with ocular melanoma will be studied by EIA, to explore their potential application in diagnosis. These studies may help to discern whether Hu-MAbs offer an advantage over MAbs of other derivation in the study of human cancers, and to learn whether any Hu-MADAs serve an important function in the tumor cell.